This application for continued support will focus on studying how protein-protein interactions among the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance protein 4 (MRP4) and inducible nitric oxide synthase (iNOS) contribute to the diarrheal symptom observed in Ulcerative Colitis (UC). Our unifying hypothesis is that CFTR, NHERF2, MRP4 and iNOS form a macromolecular complex at or near the plasma membrane in gut epithelial in UC and this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. Specifically, we proposed that iNOS is overexpressed at the plasma membrane of gut epithelia in UC, resulting in the production of sustained high level of nitric oxide (NO) which triggers NO-cGMP pathway and generates compartmentalized cGMP. Since iNOS forms a macromolecular complex with CFTR, NHERF2 and MRP4 at or near the plasma membrane, this NO-dependent compartmentalized cGMP triggers the hyperactivation of CFTR chloride channels and thus causes diarrhea phenotype. Disruption of the macromolecular complex will abolish the functional coupling of iNOS-dependent cGMP generation and CFTR Cl- channel function. The specific aims of this proposal are: Specific Aim 1. To test the hypothesis that iNOS is overexpressed in UC-affected human gut epithelia and to test whether it is coupled to CFTR and MRP4 and upregulates CFTR Cl- channel function Specific Aim 2. To test the hypothesis that iNOS, CFTR, and MRP4 form a macromolecular complex at or near the plasma membrane of gut epithelial cells and that this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. The proposed studies will not only help us better understand the mechanisms underlying the diarrhea symptom commonly observed in UC, but probably provide novel targets and methods for therapeutic interventions of the disease. Therefore, the studies proposed in this application will have clinical relevance for many individuals suffering from UC and also for those suffering from certain forms of secretory diarrheas.